Preparation for treatment of allergic patients sensitive to ragweed pollen

ABSTRACT

The preparation of this invention comprises ragweed antigens polymerized with glutaraldehyde which are composed essentially of water-soluble polymers of molecular weights from 200,000 to 20,000,000.

GRANT REFERENCE

The invention described herein was made in the course of work under agrant from the Department of Health, Education, and Welfare.

CROSS-REFERENCE

This application is a continuation of co-pending application Ser. No.63,152, filed Aug. 2, 1979, which was a division of application Ser. No.879,966, filed Feb. 21, 1978, now U.S. Pat. No. 4,180,562, which was acontinuation of application Ser. No. 788,049, filed Apr. 15, 1979, nowabandoned, which was a continuation of application Ser. No. 358,223,filed May 8, 1973, and now abandoned.

BACKGROUND AND PRIOR ART

In the United States one of the most common allergies is to ragweedpollen. Allergic patients sensitive to ragweed have been immunized byrepeated injections of ragweed antigen. The injections are givensubcutaneously. Because patients are allergic to the injected antigens,it has been necessary in clinical practice to administer a large numberof small but increasing doses over an extended period of time. Further,even with this regimen, some patients cannot tolerate the injectionsbecause of their very high sensitivity to the injected ragweed antigens.It has therefore been desired to provide a means for increasing theeffectiveness of preparations for use in treating ragweed sensitivepatients. More specifically, it has been desired to reduce theallergenicity (IgE mediated reactions) of such preparations, whilemaintaining or improving the immunogenicity (ability to induce IgGantibody). Optimally, the patient would be immunized by giving a fewlarge doses of the ragweed antigen, and the immunity would be maintainedfor several weeks or months without the need for additional injections.Heretofore, however, these objectives have not been achieved.

In the U.S. Pat. No. 3,794,630, there is disclosed a method for reactingprotein allergenic extracts with dialdehydes such as glutaraldehyde. Theresultant products which are said to be substantially water-insolubleare claimed to provide reduced allergenicity relative to the untreatedallergen. The examples disclose the preparation of such water-insolublepolymerized products from extracts of Cocksfoot pollen and timothypollen. There is no example or other reference to ragweed pollen.

SUMMARY OF INVENTION

According to the present invention, ragweed antigens, such as ragweedantigen E, are polymerized with glutaraldehyde to produce water-solublepolymers of specified molecular weight ranges. After the polymerizationreaction, the mixed polymers are fractionated in aqueous solutions. Forpurposes of the present invention, it is particularly important thatpolymers having molecular weights of less than 200,000 are removed, aswell as any residual unpolymerized ragweed antigens. Very largepolymers, such as those above 20,000,000 molecular weight, may also beremoved and this is preferred. The resulting product is thereby composedessentially of water-soluble polymers of ragweed antigens of molecularweights from 200,000 to 20,000,000, and the preparations aresubstantially free of unpolymerized ragweed antigens and polymersthereof having molecular weights of less than 200,000.

The preparations of the present invention are immunogenic, providing atleast as good and usually somewhat better immunogenicity (ability toproduce IgG antibody response) than the unpolymerized ragweed antigens.Moreover, the preparations are characterized by markedly reducedallergenicity (ability to produce allergic response), not only withrespect to unpolymerized ragweed antigens but also as compared withpreparations containing polymerized ragweed antigens of less than200,000 molecular weight. The invention therefore makes possible animproved therapy with fewer injections. For example, the required numberof injections given safely may be reduced in clinical practice to aslittle as 1/10 the injections presently required to reach maintenancedose using current therapy with unpolymerized ragweed antigens.

DETAILED DISCLOSURE

Ragweed pollen contains several antigens, the principal antigenic factorbeing identified as ragweed antigen E. The present invention may bepracticed either with mixed ragweed antigens or with isolated antigen E.For commercial purposes, the mixed antigens are preferred because theyare less expensive to prepare. In general, the ragweed pollen isextracted with other aqueous solvent to obtain the ragweed antigens,which, if desired, can be further purified. The extracted purifiedantigenic factors are then reacted with glutaraldehyde to form a mixtureof polymers. The reaction can be carried out at moderate temperatures,such as at temperatures from 20° to 30° C., using an aqueous solution ofthe glutaraldehyde to treat the ragweed antigens. A suitableconcentration of glutaraldehyde is 0.625 ml of 1:50 glutaraldehyde for10 mg of ragweed antigen. Reaction times of 3 to 5 hours are suitable.

After the polymerization has been carried out as far as desired, thepolymerization reactions may be terminated. For example, furtherpolymerization can be stopped by adding glycine, or similar reagent. Thepolymerization should not be carried to the point where any substantialamount of water insoluble polymers are produced. If some water-insolublepolymeric material is present, this should be removed, such as byfiltration. The solution of water-soluble polymers is then fractionated,such as by chromatographic separation. The adsorbents and conditions areselected so that in one separation the polymers having molecular weightsabove 20,000,000 are removed, and in a prior or subsequent separation,the polymers having molecular weights of less than 200,000 are removed.There is thereby obtained an aqueous solution of glutaraldehydepolymerized ragweed antigens having molecular weights of from 200,000 to20,000,000, and being substantially free of unpolymerized ragweedantigens, and polymers thereof having molecular weights of less than200,000. This product may be recovered in solid form by evaporation ofthe water, or by precipitation with ammonium sulfate. The solid product,being water-soluble, may then be redissolved in a suitable aqueouscarrier for subcutaneous injection (viz. buffered normal saline). Itwill be understood that the preparation should be sterilized beforeadministration, either before or after being combined with the aqueousvehicle, and that the preparation is in aqueous solution whenadministered.

Initial studies of polymerized ragweed antigens defined certain antigencharacteristics. These included the demonstration that all antigenicdeterminants of ragweed antigen E were present on the polymerizedmaterial. Two high molecular weight fractions were prepared, apolymerized ragweed antigen with molecular weights ranging approximatelybetween 200,000 and 4,000,000, designated low molecular weightpolymerized ragweed (LMW-P-RW), and a high molecular weight polymerizedragweed (HMW-P-RW) with molecular weights ranging between 4,000,000 and20,000,000. These two preparations produced immune responses in guineapigs and rabbits. Further studies were done to evaluate thecharacteristics of the polymerized ragweed antigens including thosewhich might apply to potential therapeutic usefulness in therapy ofhuman patients with ragweed pollenosis. The procedures are illustratedby the following examples.

EXAMPLE 1

Dwarf ragweed (Ambrosia elatior) is fractionated by the method of Kingand Norman, Biochemistry, 3:458 (1964). The fraction IV obtained byammonium sulfate precipitation, diethylaminoethyl cellulose and Sephadexchromatography was further fractionated through triethylaminoethylcellulose and Fraction IVC containing nearly 100 percent Antigen E wasremoved and used for certain studies. AgE has a molecular weight ofabout 38,000. The remainder of Fraction IV, containing Fractions IVA,IVB, and IVD were combined. This pool of Fraction IV minus IVC wascalculated to contain about 60 percent ragweed antigen E and was usedfor polymerization by glutaraldehyde treatment. Fraction IVC isdesignated AgE and Fraction IV minus IVC is designated RW. RW was latershown to be about 30 percent AgE by immunoassay.

All preparations of RW were evaluated by measuring optical density at280 nm with a Beckman DU spectrophotometer. One O.D. unit was estimatedto be approximately 1 mg of protein per ml and expressed as such forsubsequent immunologic studies.

EXAMPLE 2

RW, obtained by the method of Example 1, comprising mixed ragweedantigens with a content of about 30% antigen E, was treated withglutaraldehyde using the method of Sachs and Winn, Immunochemistry,7:581 (1970). Polymerization was done at room temperature for 4 hours.For 10 mg of RW, 0.625 ml of 1:50 glutaraldehyde was used. 10 to 30 mgof the RW protein was used for glutaraldehyde treatment on each occasionthat polymerized RW (P-RW) was prepared. Preparations of P-RW werefractionated using Sephadex or Sepharose (Pharmacia Fine Chemicals AB,Uppsala, Sweden). Sephadex G-200, or Sepharose 6B, 4B or 2B were usedfor various fractionations using 40×2.5 cm columns. The Sephadex andSepharose columns and protein solutions were equilibrated with a 0.1 MTRIS, 0.15 M NaCl buffer, pH 7.9. The protein content of the eluates wasdetermined by the optical density at 280 nm using a Beckman DUspectrophotometer. Dextran Blue 2000 was used as a molecular marker invarious columns to aid in determination of excluded and includedfractions. Polymerized material having a molecular weight in excess of200,000 was thereby obtained.

When RW is treated with glutaraldehyde and the resultant P-RW wasfractionated through a Sephadex G-200 column, the major portion of theglutaraldehyde treated RW protein appears in a defined peak. Thismaterial in the exclusion volume of the Sephadex G-200 has a molecularweight in excess of 200,000. Further fractionation of the polymerizedmaterial using a column of Sepharose 6B gave two protein fractions.These were a sharp peak containing protein in the exclusion volume ofSepharose 6B and a broad shoulder in the inclusion volume. The sharppeak is considered to be composed of polymerized ragweed with amolecular weight in excess of 4,000,000 termed high molecular weightpolymerized ragweed (HMW-P-RW) (Pool I). The second, diffuse proteinfraction is considered to have a spectrum of material with molecularweights ranging from 200,000 to 4,000,000 (LMW-P-RW) (Pool II). WhenPool I was fractionated through a Sepharose 4B column, 2 peaks wereobtained. The first peak in the exclusion volume of Sepharose 4B, wasconsidered to have a molecular weight in excess of 20,000,000 and thesecond peak in the inclusion volume of Sepharose 4B, was considered tohave a range of molecular weights from 4,000,000 to 20,000,000. BothHMW-P-RW and LMW-P-RW were entirely water soluble.

EXAMPLE 3

Subsequent to the above studies showing the various ranges of molecularweights of polymerized ragweed resulting from gluaraldehyde treatment ofragweed, a standard procedure for evaluation of the polymerized materialwas developed. These studies used the addition of ¹²⁵ I AgE to RW inorder to examine the effect of glutaraldehyde treatment on a majorantigen of RW. The ¹²⁵ I AgE is not, of course used in the materialsprepared for human use. The RW with added ¹²⁵ I AgE was treated withglutaraldehyde and fractionated through Sepharose 6B. The radioactiveprotein (¹²⁵ I AgE) included in the Sepharose 6B had molecular weightranges less than 4,000,000. The included Sepharose 6B fractions werefractionated through Sephadex G-200 and the excluded fractions had amolecular weight of more than 200,000. These studies showed the ¹²⁵ IAgE in RW was polymerized and the polymers of LMW-P-RW had molecularweight ranges of 200,000 to 4,000,000. The excluded fractions fromSepharose 6B (molecular weight more than 4,000,000) were fractionatedthrough Sepharose 4B. The ¹²⁵ I AgE was found in the included fractions.These included fractions, termed HMW-P-RW, had a molecular weight rangeof 4,000,000 to 20,000,000. These procedures were done because of thevariable degree of polymerization which could occur during thepreparation of P-RW and to define the polymerization process with amajor radiolabelled antigen.

The preparations of RW material polymerized with glutaraldehydetreatment showed a wide range of molecular weights as demonstrated bythe elution patterns of the Sepharose 6B, 4B and Sephadex G-200fractionations. Although slight variability in the patterns was observedwith different batches, there was good reproducibility in more than 5individual polymerization experiments. The yield of polymerized materialwas over ninety percent as estimated from the optical density yield ofthe polymerized materials and the polymerized materials remained solubleduring the course of these experiments. The products were water-solublepolymers ranging in molecular weights from below 200,000 to above20,000,000. Previous reports of glutaraldehyde treatment of otherproteins (Sachs et al., Immunochemistry, 8:58 (1970) demonstrated thatless than one percent of the proteins were uncoupled. In evaluations ofRW polymer stability, the polymer was shown to be stable during standardstorage conditions up to 6 months.

The RW treated with glutaraldehyde retained its antigenicity and reactedwith all antibody in antiserum prepared against unpolymerized RW.Although the polymerized ragweed preparations could react with allantibody against RW antigen in an antiserum, as demonstrated byneutralization experiments, increasing concentrations of the polymerizedpreparations were required to accomplish this in proportion to themolecular size of the polymers.

The antibody responses in guinea pigs immunized with RW, LMW-P-RW andHMW-P-RW demonstrated that all preparations produced antibody responsesbut these were not evident until after tertiary immunization even with atechnique as sensitive as the ammonium sulfate isotope labeled antigentechnique used for detection. This lack of response to primary andsecondary stimulation was not unexpected as good antibody responses toragweed antigens generally requires repeated antigen administration. Theresults of antibody responses in guinea pigs to the 3 RW materialsdemonstrated that the polymerized materials are at least as antigenic inthis species as RW and that the LMW-P-RW is probably the most antigenicof the 3 molecular species.

Results of immunization experiments with rabbits were similar and alsosuggests that immune response to injection with LMW-P-RW is at least asgood as, and probably superior, to the RW or HMW-P-RW.

Glutaraldehyde polymerized preparations of ragweed with a significantportion of antigen E, the major antigen of ragweed pollen, have certaincharacteristics. These include: complete reactivity of the polymerizedragweed with antibody against the monomeric form, no signficantformation or exposure of new antigenic determinants duringpolymerization, antigenicity which is at least that of the nativeantigen E, reactivity with IgE antibody, and a marked diminution ofallergic reactivity on a weight basis, as demonstrated by histaminerelease from cells from allergic humans in vitro and allergic skinreactions to RW sensitive humans. The results suggest the potentialusefulness of these preparations for human allergen immunotherapy withantigen E and possibly other purified inhalent antigens. This potentialuse is based on the persistent and probably enhanced antigencity ofpolymerized ragweed preparations combined with their diminishedreactivity with IgE sensitized mast cells and their diminished rate ofabsorption from locally injected sites as compared with the nativeantigen E monomer.

EXAMPLE 4

Pollens were obtained from Hollister-Stier Laboratories (Spokane,Washington). Giant and short ragweed pollens were used. These pollenswere defatted with ethyl ether several times. The defatted pollen grainswere extracted with isotonic phosphate buffered saline, pH 7.2 (PBS) at4° C. for 24 hours. The preparation was centrifuged at 17,000×G for 1 hrand the precipitate was discarded. Proteins in the crude extract wereprecipitated with ammonium sulfate at 90 percent saturation level.Resulting precipitate was dissolved in PBS, and dialyzed at 4° C.against PBS until free of ammonium sulfate. Further purification ofextract was done by chromatography on a Sephadex G-15 column. Theexcluded portion of Sephadex G-15 was the monomer ragweed (MRW).

For each 10 mg MRS, 0.625 ml 1:50 glutaraldehyde (25% grade II, SigmaChemical Company) was added. The polymerization of MRW was carried outat room temperature for 4 hours using a magnetic stirrer. Then glycinewas added to stop further polymerization. The glutaraldehyde treatedmixture was fractionated on a Sepharose 4B (Pharmacia) column to removepolymers above 20,000,000 daltons and the fractions included by theSepharose 4B column were combined. The polymerized ragweed (PRW) ofbelow 20,000,000 daltons was placed in a dialysis bag and concentratedby evaporation to 4 cc and dialyzed free of excess glutaraldehyde andglycine. The PRW was then applied to a Sephadex G-200 (Pharmacia)column. The fractions excluded (above 200,000 daltons) or included(below 200,000 daltons) by the Sephadex G-200 were combined and storedat -20° C. until use. Preparation I thus prepared contained polymerswithin the range from 200,000 to 20,000,000, while Preparation IIcontained polymers below 200,000 molecular weight. Before human use,they were dialyzed for 48 hrs against phosphate buffered 0.15 M NaCl, pH7.35, containing 0.5% phenol.

RW preparations were sterilized by filtration using a filter unit (0.20micron plain membrane, Nalge, Sybron Corp., N.Y.). They were culturedfor sterility by the microbiology laboratories of Northwestern MemorialHospital at 48 hrs and following 2 weeks of culture. Preparations wereproved to be sterile before use in the human studies reported below.

Cutaneous titration (human). Three ragweed sensitive subjects weretested by intracutaneous injection of 0.02 ml of serial 10-folddilutions of each preparation. Reactions were graded 1 to 4 pluspositive and the endpoint titration was the highest dilution giving awheel and erythema reaction. The results are summarized in the follwingTable A.

                  TABLE A                                                         ______________________________________                                                       Allergenic Responses of                                        Polymerized    Three Ragweed Sensitive Subjects                               Ragweed Antigens                                                                             (endpoint dilution titers*)                                    (molecular weight)                                                                           Subject A Subject B Subject C                                  ______________________________________                                        I.   200,000-20,000,000                                                                          10.sup.-2 negative                                                                              negative                                 II.  below 200,000 10.sup.-8 10.sup.-8                                                                             10.sup.-5                                III. combined I & II                                                                             10.sup.-3 10.sup.-5                                                                             10.sup.-1                                ______________________________________                                         *The number recorded is the highest serial 10fold dilution giving a           positive wheal and erythema reaction.                                    

The foregoing results demonstrate that ragweed allergens treated withglutaraldehyde have dramatically reduced allergenicity in the molecularweight range of 200,000 to 20,000,000. On the other hand, allergenicityis retained in the lower molecular weight polymers (below 200,000) eventhough they have been subjected to the identical treatment withglutaraldehyde. The difference in degree of allergenicity as determinedby cutaneous endpoint titration varied from at least 10⁵ to 10⁸. Thereduced allergenicity of Preparation I was not at the expense ofimmunogenicity, as has already been demonstrated.

EXAMPLE 5

Using a similar procedure of that described in Example 4 ragweedantigens (King's IV-IVC) were polymerized with glutaraldehyde, andsubjected to chromatographic separation to prepare a polymerized productcontaining polymers within the molecular weight range from 200,000 to20,000,000. This preparation (PRW) was compared with the unpolymerizedragweed antigens (MRW) with respect to the degree of allergenicity.Three ragweed sensitive human subjects were tested by intracutaneousinjection of 0.02 ml of serial 10-fold dilutions of both PRW and MRW.Reactions were graded 1 to 4 plus positive, and the endpoint titrationwas the highest dilution given a wheel and erythema reaction. Theresults are reported below in Table B and show that in this experimentthe polymer was 100 to 1000 fold less allergenic.

                  TABLE B                                                         ______________________________________                                        COMPARATIVE ALLERGENICITY OF POLYMERIZED                                      AND UNPOLYMERIZED RAGWEED ANTIGENS                                            Concen-                                                                       tration of                                                                    RW tested                                                                             Subject 1   Subject 2    Subject 3                                    (ug/ml)*                                                                              MRW     PRW     MRW    PRW   MRW   PRW                                ______________________________________                                        50      ND      ND      ND     ND    ND    +++                                5       ND      +++     ND     ND    ND    -                                  0.5     ND      -       ND     +++   +++   -                                  0.05    +++     -       ++++   -     ++    -                                  0.005   ++      -       ++     -     -     -                                  0.0005  -       -       -      -     -     -                                  ______________________________________                                         *based on optical density at 280 nm using a Gilford 250 spectrophotometer     Abbreviations:                                                                RW, ragweed antigen;                                                          MRW, unpolymerized ragweed antigens;                                          PRW, polymerized ragweed (200,000-20,000,000 daltons);                        ND, not done.                                                            

EXAMPLE 6

It can be demonstrated that RW antigens are not only incorporated in theRW Polymers of this invention, but once incorporated, that they are alsoeffective immunogens. The following study is illustrative. Serum samplesfrom 2 patients immunized with RW polymer were studied. Pure RW antigensused included Antigen E (described above), Ra3 and Antigen K, the lattertwo being obtained from the National Institutes of Health. The resultsare shown in Table C and demonstrate significant IgG antibody responsesagainst the available RW antigens.

                  TABLE C                                                         ______________________________________                                        Immune response in ragweed allergic humans treated with                       polymerized RW                                                                Percent increase.sup.+ in IgG antibody* activity                              Subject Crude Ragweed AgE      AgK    Ra3                                     ______________________________________                                        1       330           390       200    70                                     2       700           800      300    4700                                    ______________________________________                                         ##STR1##                                                                      *Determined by solid phase radioimmunoassay using polystyrene tubes      

EXAMPLE 7

(a) Clinical trials with glutaraldehyde polymerized RW (molecular weight200,000 to 4,000,000) compared with monomeric RW showed that polymerizedragweed antigens was as effective as monomeric RW in control of symptomsragweed allergic rhinitis as evaluated by symptom score indices. Thepolymerized RW produced fewer allergic reactions during the immunizationprocess.

(b) Clinical trials with glutaraldehyde polymerized RW (Molecular weight200,000 to 20,000,000) compared with monomer RW showed that the polymerRW was as effective in control of ragweed allergic rhinitis as monomer.IgG response to AgE was similar. Allergic reactions to the preparationsduring immunization treatment were markedly reduced in the polymergroup.

These studies showed that polymer RW (molecular weight 200,000 to20,000,000) was at least as effective in treatment of ragweed allergicrhinitis as monomeric RW.

EXAMPLE 8

Immunization with the polymerized RW of this invention significantlyreduced allergic reactions during treatment. Consequently, as shown bythe following studies, the number of injections of polymer RW can bemarkedly reduced, as compared with currently available ragweedimmunization therapy. The results are reported in Table D.

                  TABLE D                                                         ______________________________________                                        Comparison of safe dosage schedules for ragweed immuniza-                     tion using currently available therapy with aqueous (monomeric)               ragweed and polymeric ragweed. -                                                            Aqueous (monomeric)                                                                           polymer                                         Ragweed       Ragweed                                                         ______________________________________                                        Starting dose.sup.a                                                                         2.5       P.N.U..sup.b                                                                            500  P.N.U.                                 Maintenance dose.sup.a                                                                      2500.0    P.N.U.    2500 P.N.U.                                 Number of injections to                                                       reach maintenance dose.sup.a                                                                30-50               3-5.sup.c                                   ______________________________________                                         .sup.a Starting and maintenance doses may vary with practicing allergist      as may the rate of progression of dose. These are procedures which usuall     produce no local or systemic allergic reactions. All patients do not          tolerate this schedule.                                                       .sup.b P.N.U.: Protein nitrogen units. One P.N.U. contains 10 ng protein      nitrogen. P.N.U. is a commonly used expression of allergen potency. A 1:5     weight per volume extract of ragweed pollen contains approximately 10,000     P.N.U.                                                                        .sup.c 25 typical ragweed allergic patients have received this therapy.  

We claim:
 1. A mixture of polymers of ragweed antigen E, said polymermixture being the product of the reaction of antigen E withglutaraldehyde, all of the polymers of said mixture being water solubleand comprising polymers of molecular weights both above and below200,000.
 2. The process of preparing a preparation for treatment ofpatients sensitive to pollen, comprising reacting ragweed antigen withglutaraldehyde to form a water soluble polymer mixture containingpolymers both above and below 200,000 molecular weight, andfractionating said polymer mixture to obtain a preparation of molecularweight above 200,000 substantially free of polymers of molecular weightbelow 200,000.